ELISA common problems and countermeasures


possible reason


No color

The time of reagent incubation did not follow the instructions.

Determine whether the time for biotinylated antibody, linked HRP reagent or streptavidin-hrp is appropriate.

Mix reagents in different kits or batches.

Recheck the label of the reagent to ensure that all components belong to the reagent kit being used. Do not mix reagents in different kits or batches.

Missed enzyme

Check the operation process, be careful not to miss

hrp enzyme contaminates sodium azide

Use freshly prepared reagents and ban sodium azide

There is a problem with the standard (if there is a signal in the specimen hole)

Check the preparation process of standard products according to the instructions

Use 1 new standard

A container is not cleaned, leaving inactivated enzyme material

Use the inner container of the kit as much as possible, and the other must be clean and reliable

Prepare / re-dissolve antibodies using serum-containing buffer

Reconfirm the selected reagent

. Missing addition of developer a or b

Observe the liquid level in the hole after adding the developer

Incorrect reagent preparation / use

Redo the experiment; follow the instructions strictly, and read the label clearly before each preparation and use.

Buffer contamination

Prepare fresh buffer

Weak color rendering

Products beyond the expiration date may produce a weak signal.

Check the expiration date of the product

Wrong volume and time of reagent added

Make sure that the volume of each reagent used is correct and the addition time is appropriate.

Reagents and samples are not balanced before use

Allow reagents and samples to equilibrate at room temperature for about 10 minutes before use

Shortening the incubation time can weaken the signal of the experiment.

Check the time of incubation.

Incubate the microtiter plate in an environment with varying temperatures.

Determine the incubation temperature and avoid temperature changes.

Contaminated reagents used

Check if the reagent is contaminated.

Standards / specimen preparation methods are not standardized

Check the preparation of the standard / specimen. The standard should be reconstituted and diluted according to the instructions. Specimens stored at low temperatures avoid repeated freezing and thawing, and the use of hemolytic specimens is strictly prohibited. The sample is preserved with nan3, which inhibits the enzyme reaction

Irregular substrate preparation

Check the preparation of the substrate, such as the correct volume and proper mixing.

Improper detection time

Whether to test within the specified time.

The instrument settings are incorrect and the filters do not match.

Whether the instrument is set correctly and whether the filter selection is consistent.

High background (background)

Washing operation is not standardized

Inadequate plate washing is often seen when using manual plate washing. It is best to use a plate washer, or use a bottle wash. Each well should be completely filled with washing buffer and should be poured quickly. If a plate washer is used, it should be calibrated and set to a volume sufficient to fill each well. The inside of the board should not touch the device.

Check if there is any residual washing liquid in each well or the volume of sample added per well is accurate. Add 30 seconds of soaking between plate washes.

Incubation temperature and time are inappropriate in the experiment

Determine whether the incubation temperature and time for each experimental step are appropriate

Enzyme added too much

Check the pipette adjustment amount before adding enzyme.

Check the dilution, if necessary, do potency determination test.

Incomplete closure

Check the calculated amount of blocking solution; extend the blocking time.

Interfering substances in specimens or standards

Make appropriate comparisons

The developer is exposed to light for a long time or is contaminated

Developers a and b should be removed from the refrigerator 10 minutes before use

The entire batch of samples is placed for too long, and the samples are contaminated

Samples should be kept fresh or stored at low temperature to prevent contamination

Buffer contamination

Prepare fresh buffer

Reusable tips, not washed or incompletely disinfected and used for adding enzymes or color reagents

Tips for single use as much as possible

Too many signals:

All the boards become regular blue

Insufficient washing / washing steps were missed-unbound peroxidase remained.

It is best to use a washing machine to fully wash

Check if there is any residual lotion in the hole or if the sample volume is accurate.

Substrate solution mixed too early and turned blue

The timing of substrate mixing should be controlled and used immediately

Too many enzyme conjugates

Check dilution and perform potency determination if necessary

The sealing film or reagent container is reused, resulting in the residual hrp, which makes the tmb substrate produce a non-specific blue.

Use a new sealing film and use different reagent containers for each step

Contaminated metals or hrp in the buffer

Prepare fresh buffer

High cv value (cv: coefficient of variation), flower plate

Inadequate operation or insufficient washing

Wash the plates, add samples and develop colors according to the instructions. Washing the board is particularly important, as mentioned above

Dry board appears, no sealing film is used, and the sealing film is reused

Make sure that the microplate should be kept moist between every two steps.

Use sealing film to seal, pay attention to using new sealing film every step.

The package is uneven due to operational errors or poor board quality (uneven bonding).

Do not add other proteins to the diluted pbs

Check the volume and time of the coating and blocking solution and the method of reagent addition.

Check the microplates used, use elisa plates (do not use tissue culture plates)

The pipette is inaccurate and the tip is reused.

Check and calibrate the pipette. The tip must be changed every time a sample is taken. Review the steps of adding specimens to ensure the accuracy of each sample addition, and ensure that the sucked liquid is sucked and discharged according to the set volume. Pay attention to check the suction tip during continuous sampling to ensure the volume of the added liquid.

Insufficient centrifugation of the sample, clotting or residual cell components in the reaction well

Specimens are fully centrifuged at 3000rpm for more than 6 minutes. The use of coagulated (hemolytic) specimens is strictly prohibited

Buffer contamination

Prepare fresh buffer

Standard curve is available, but the difference between the two points is poor (low or flat curve)

Insufficient enzyme conjugate

Check dilution and perform potency determination if necessary

Capture antibody does not bind well to the plate

Check the microplates used, use elisa plates (do not use tissue culture plates)

Do not add other proteins to the diluted pbs

Insufficient detection antibody

Check dilution and perform potency determination if necessary

Insufficient color development on the board

Extended substrate incubation experiment

Use recommended brand substrate solutions

Careless operation

Review the elisa operation process to eliminate any unauthorized modification procedures.

Incorrect calculation of standard curve dilution

Check the calculation and prepare a new standard curve

The standard curve is good, but there is no expected positive signal

No corresponding cytokines in the specimen

Use internal control

Repeat the experiment and reconsider the corresponding parameters of the experiment

Specimen matrix detection

Dilute the specimen at least 1: 2, or perform serial dilution to observe its recovery

The standard curve is very good, but the interpretation value of the specimen is very high

The level of cytokines contained in the specimen exceeds the detection range

Dilute the specimen and experiment again

When using hrp enzyme conjugate, tmb substrate and stop solution will turn green

Insufficient color development of reagent in well

Gently shake the board

Edge effect

Uneven working temperature

Avoid incubating the microtiter plate in a changing temperature environment


Discontinuity during the experiment

The entire experiment should be operated continuously: prepare all standards and specimens properly before the experiment

The reagent is not equilibrated to room temperature according to the instructions

Before all reagents are added to the wells, make sure they are equilibrated to room temperature, unless otherwise stated in the instructions.

Is it possible to change the experimental operation provided by the kit


In order to ensure the highest sensitivity and specificity, general manufacturers have optimized the kits. To ensure the standardization of the experiments of each kit, they should follow the instructions.

Can the reagents in different kits be mixed?

Not allowed. Most reagents are specific in each batch of kits, if there is a problem, please contact the manufacturer or agent.

Whether the volume of the specimen can be increased or decreased.

The volume of the specimens to be added in the commercial kit is optimized, and it should be operated according to the instructions. It is not recommended to change the volume of the specimens added.

Can I redefine the point of my own standard curve?

can. There are recommended dilutions of the standard when preparing the standard curve in the instruction manual, which can change the dilution factor and increase the point of the curve, but it must be within the detection range. Invalid.

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